Abstract Significant progress has been made in recent years in osteoarthritis (OA) research, including the generation of mouse models, including genetic and surgically-induced OA mouse models that show an OA-like phenotype. One feature that is common to these mouse models of OA is the up-regulation of Runx2. Because Runx2 is a key transcription factor directly regulating the expression of genes encoding for matrix degradation enzymes, such as Mmp9, Mmp13 and Adamts5, this up-regulation leads to the increase in expression of these genes. The key questions have been raised. 1) Is Runx2 a central molecule mediating OA development in joint tissue? 2) Could manipulation of Runx2 expression be used to treat OA disease? miRNAs are endogenous non- coding RNAs and play important roles in regulation of RNA stability and protein expression. Previous studies by our lab have shown that miR-204/-211, two homologous miRNAs, bind Runx2 and regulate Runx2 expression in mesenchymal stem cells (MSCs). To determine the function of these miRNAs in Runx2 regulation and in OA development, we have recently created miR-204 and miR-211 floxed alleles and further generated miR-204/-211 double KO mice (dmiRPrx1 and dmiRAgc1ER) by breeding miR-204flox/flox and miR- 211flox/flox mice with Prx1-Cre (targeting limb MSCs) or Agc1-CreER (targeting adult articular chondrocytes) transgenic mice to delete these miRNAs. A severe OA-like phenotype was observed in dmiRPrx1 and dmiRAgc1ER KO mice and entire knee joint tissues were affected by miR-204/-211 deletion, including severe loss of articular cartilage, subchondral sclerosis, chondrophyte/osteophyte formation, and synovial hyperplasia. Our findings suggest that miR-204 and miR-211 are key regulators in maintaining joint tissue homeostasis and could be served as critical targets for OA treatment. The underlying hypothesis of this project is that miR- 204 and miR-211 are key regulators of Runx2 expression and play critical roles in maintaining joint tissue homeostasis. Two specific aims have been proposed to test this hypothesis. In Aim 1, we will fully characterize the longitudinal effects of miR-204/-211 deletion on OA development in dmiRAgc1ER KO mice and determine if deletion of Runx2 in chondrocytes will reverse OA phenotype observed in dmiRAgc1ER KO mice. In Aim 2, we will investigate changes in miR-204/-211 expression in OA samples and determine if in vivo administration of miR-204 will prevent OA development or decelerate OA progression in surgically-induced OA mouse model. These proposed studies will provide novel insights into mechanisms of OA development and will help the development of new strategies for OA treatment.